Technologies are needed that enable comprehensive biomarker and target discovery for detection, prognosis, patient stratification, and therapeutics. Methods of detecting biomolecules (e.g., polypeptides, nucleic acids, polysaccharides, and lipids) are widely used in a variety of fields including, e.g., proteomics and genomics. Detection, analysis, and identification of biomolecules can play an important role in the understanding of the biology of organisms and the causes of disease. (See, e.g., Pandey et al., Nature, 405: 837-846 (2000).
In particular, oxidation of biomolecules including nucleic acids, lipids, and proteins has been implicated in several diseases including, e.g., Alzheimer's disease, acute respiratory distress syndrome, amyotrophic lateral sclerosis, cataractogenesis, chronic lung disease, bronchopulmonary dysplasia, chronic renal failure, chronic uremia, cystic fibrosis, dementia, diabetes, ischemia-reperfusion, Parkinson's diease, preeclampsia, psoriasis, rheumatoid arthritis, juvenile and chronic arthritis, severe sepsis, systemic amyloidosis, and varicocele (see, e.g., Butterfield, Brain Res. 1000:1-7 (2004); Butterfield and Castegna, Amino Acids 25:419-425 (2003); Dalle-Donne et al., Trends Mol. Med. 9(4):169-176 (2003); Himmelfarb and McMonagle, Kidney Int'l. 60:358-363 (2001); and Odetti et al., Acta Diabetol 36:179-183 (1999). Often, oxidation patterns of biomolecules change (e.g., increase or decrease) with disease progression or regression. In addition, oxidation patterns (i.e., the identity of specific oxidized sites) changes with disease progression or gresssion. For example, as the severity of neurodegenerative disorders such as Alzheimer's disease increases, the levels of oxidized creatinine Kinase BB, glutamine synthase, ubiquitin carboxy-terminal hydrolase L-1, and dihydropyrimidinease related protein 2 increases (see, e.g., Butterfield and Castegna, Amino Acids 25:419-425 (2003)). Conversely, as the severity of a neurodegenerative disease is diminished, e.g., in conjunction with pharmaceutical therapy, the levels of oxidized proteins decreases.
A number of different technologies have been developed to separate, analyze and identify biomolecules such as proteins. For example, efforts to identify oxidized proteins and to map oxidized proteins using monoclonal antibodies, two dimensional gel electrophoresis, HPLC, and mass spectrometry have been described (see, e.g., Butterfield, Brain Res. 1000:1-7 (2004); Butterfield and Castegna, Amino Acids 25:419-425 (2003); Himmelfarb and McMonagle, Kidney Int'l. 60:358-363 (2001); and Odetti et al., Acta Diabetol 36:179-183 (1999)). However, many of these methods are inconvenient for simulataneous analysis of multiple samples.
Typically, identification by mass spectrometry (MS) involves analysis of isolated proteins or peptide fragments, followed by mapping or tandem MS to obtain sequence information. One strategy that has been used to differentiate the resulting spectra involves tagging the proteins with reagents having different masses (“mass tags”). The most predominant mass tags are based on the mass difference of the isotopes hydrogen and deuterium. The isotopically distinct mass tags are referred to as Isotope-Coded Affinity Tags (ICAT), and their use allows a number of different samples to be analyzed at the same time and directly compared. See, e.g., Ranish et al. Nature Genet. 33: 349-355 (2003); Zhou et al., Nature Biotechnol. 19: 512-515 (2002); Gygi et al., J. Proteome Res. 1: 47-54 (2002); Gygi et al., Nature Biotechnol. 17: 994-998 (1999); Gygi and Aebersold, Curr. Opin. Chem. Biol. 4: 489-494 (2000); Aebersold and Mann, Nature 422: 198-207 (2003); Patterson and Aebersold, Nature Genetics Suppl. 33: 311-323 (2003); and Tao and Aebersold, Curr. Opin. Biotechnol. 14: 110-118 (2003); and WO 00/11208. The reagent consists of biotin for affinity selection, a linker that contains light (hydrogen) or heavy (deuterium) isotopes of hydrogen for mass tagging, and a Cys-reactive group (iodoacetamide) to derivatize proteins. Differential labeling involves using two isotopic reagents for two samples in comparative profiling. Samples are mixed following the ICAT derivatization step and proteolyzed together. The tagged peptides are affinity purified using an avidin column, and analyzed by mass spectrometry. The ratio of mass peak amplitude of peptides from proteins differentially labeled with heavy and light mass tags gives a measure of the relative amounts of each protein. The ICAT method, using a heavy reagent and a light reagent, is limited to differential analysis of two samples.
ICAT has a number of shortcomings. First, ICAT utilizes only two different masses (light and heavy). Thus, the method is limited to applications that require comparisons of only two states. Second, cysteine (Cys) is an amino acid of low abundance (about 2.2%). Moreover, many cells contain endogenously biotinylated proteins, the proteolyzed fragments of which are immobilized by the affinity column. Of particular note is the tendency of the deuterated and non-deuterated probes to elute differentially, giving rise to more than one peak. Finally, the biotinylated tags have a tendency to fragment during mass spectrometric analysis.
In view of the shortcomings of tagging methodologies as presently practiced, there is a need in the art for methods of detecting, analyzing, and identifying oxidized biomolecules in a sample, including those present only in small quantities. A method that was more versatile and robust than those methods based upon ICAT would overcome current limitations in biomolecule analysis. The present invention provides such a method.